RP534 THE OXIDATION OF ALPHA AND BETA GLUCOSE AND A STUDY OF THE ISOMERIC FORMS OF THE SUGAR IN SOLUTION By Horace S. Isbell and Ward Pigman ABSTRACT The method of bromine oxidation in the presence of barium carbonate has been applied to the measurement of the rates at which the various forms of d-glucose react with bromine water. Under the conditions described /3-d-glucose is oxidized about thirty-five times as fast as a-d-glucose. The delta lactone of gluconic acid is the predominating product in both cases, and it is formed without the inter- mediate production of gluconic acid. This indicates that a- and ,8-d-glucose con- tain the 1, 5 or amylene oxide ring. The oxidation of an equilibrium solution of d-glucose progresses rapidly, until all of the beta glucose present in the solution is used up, and then more slowly as the oxidation of the alpha form is continued. The rate of oxidation indicates that the equilibrium solution contains about 64 per cent beta and 36 per cent alpha glucose. There can be little doubt but that the equilibrium of glucose is more complex than the simple equilibrium between the alpha and beta forms, but the absence of substantial amounts of the free acid and the gamma lactone in the product of oxidation is evidence that the equilibrium solution of the sugar does not contain large proportions of the free aldehyde and of the gamma form. It is believed that the application of the methods of investigation described in this report will yield much useful informa- tion as to the composition of sugar solutions and the changes which occur during the mutarotation reaction. CONTENTS Page I. Introduction 337 II. Oxidation of alpha and beta glucose 338 III. Reaction rates 344 IV. Experimental details 352 1. Oxidation of /3-^-glucose to gluconic delta lactone 352 2. Oxidation of a-d-glucose to gluconic delta lactone 354 3. Measurement of reaction rates 355 4. Mutarotation of a-d-glucose atO° C 356 V. Applications of method 356 I. INTRODUCTION Inasmuch as solutions of reducing sugars presumably contain a number of isomeric forms of the sugar in equilibrium, it is desirable to ascertain which isomer is the active reductant in any oxidation. Previous investigators have assumed that solutions of the aldoses contain small quantities of the aldehyde form of the sugars and that the monocarboxylic acid is derived by the oxidation of this free alde- 2 i hyde. It was shown by Isbell and Hudson that the optical rotation 1 of a buffered glucose solution, upon bromine oxidation, rises to a point which corresponds with the rotation of the delta lactone, and then the rotation decreases to a minimum value at a rate approximately 1 This paper was presented at the meeting of the American Chemical Society, New Orleans, La., March 29 1932 » Isbell and Hudson, B. S. Jour. Research, vol. 8 (RP418), p. 327, 1932. 337 338 Bureau of Standards Journal of Research [Vol. w the same as the hydrolysis of the delta lactone to the corresponding 3 acid. Subsequently it was shown by Isbell that the free acid is not formed in the course of the oxidation of the aldoses with bromine water by conducting the reaction in the presence of barium carbonate. The latter neutralizes the free acid but does not react with the lactone, thereby differentiating between the two substances. This method, as well as the changes in optical rotation and acidity, prove that delta lactones are obtained prior to the acids from a- and /3-d-mannose, a- and /3-Z-rhamnose, a- and /3-lactose, and /3-maltose. Therefore, it is apparent that the normal aldoses, at least in slightly acid solution, do not yield acids by bromine oxidation and consequently there is no foundation for the heretofore accepted concept that the reaction takes place through the straight-chain aldehyde form of the sugar. Since, in strongly acid solutions, the oxidations are slow and the oxidation products are rapidly converted into equilibrium mixtures containing the free acid, the delta and gamma lactones, extreme care must be exercised in the interpretation of the results thus obtained. In a paper entitled " A Possible Source of Error in Determining the Constitution of Di and Polysaccharides," published prior to the publication of Isbell and Hudson, 4 on "The Course of the Oxidation of the Aldose Sugars with Bromine Water," but subsequent to the actual work done by Isbell and Hudson, Irvine and McGlynn 6 make the following statement: "Crystalline 2, 3, 6 trimethyl glucose if oxidized with bromine water under conditions which permit the accumulation of hydrogen bromide yields directly the gamma lactone of 2, 3, 6 trimethyl gluconic acid as the main product—but when the sugar is oxidized by the same reagent in the absence of halogen acid, the delta lactone is produced in preponderating amount. " However, as pointed out by Hirst, 6 their experimental work does not show that the gamma lactone is formed directly because the reaction in acid solution was allowed to continue eight hours, which is ample time, especially in the presence of hydrobromic acid, for the primary oxidation product to be rearranged into the gamma lactone. Irvine and McGlynn's experimental procedure, also, did not show that the delta lactone was the primary oxidation product of the bromine oxidation in the absence of halogen acid, because they allowed the reaction to proceed for eight hours in the presence of calcium car- bonate. In eight hours the lactone would have been hydrolyzed, for the most part, to give the free acid, which would have reacted in turn with calcium carbonate to give the calcium salt. Irvine and McGlynn state: "The calcium salts in solution were decomposed by the addition of the required amount of oxalic acid, the product extracted with chloroform and isolated in the usual manner." By this procedure a mixture containing the delta and gamma lactones would have been obtained regardless of the nature of the initial oxidation product. II. OXIDATION OF ALPHA AND BETA GLUCOSE The method, which has been reported in previous publications, 7 consists in oxidizing the sugar with bromine water in a solution satu- rated with carbon dioxide and buffered with a suspension of barium « Isbell, B. S. Jour. Research, vol. 8 (RP441), p. 615, 1932. 4 See footnote 2, p. 337. 6 Irvine and McGlynn, J. Am. Chem. Soc, vol. 64, p. 359, 1932. • Hirst, J. Am. Chem. Soc, vol. 54, p. 2559, 1932. ' Isbell, see footnote 3, p. 338; also J. Am. Chem. Soc, vol. 54, p. 1692, 1932. — — Isbell 1 Pigmanj Oxidation oj Alpha and Beta Glucose 339 carbonate. The barium carbonate prevents the solution from becoming strongly acid, and consequently the oxidation is rapid, while the mutarotation reaction is slow. In the present instance the crystalline sugar or the equilibrium mixture was added to the buffered bromine solution and from time to time the oxidation was stopped by removing the bromine through reaction with an unsaturated oil or hydrocarbon ; the excess buffer (barium carbonate) was then separated by filtration, leaving a solution containing the oxidation product, any unoxidized sugar, barium bromide, and traces of barium bicarbonate. Any free gluconic acid, formed either in the course of the oxidation or by the hydrolysis of the lactone, would be neutralized by the barium carbonate giving barium gluconate, while the unchanged lactone would exist in the solution as such. The amount of the lactone in the solution was determined by titration; the unoxidized sugar was determined by reduction of Benedict's solution; and the amount of gluconic acid neutralized by the barium carbonate was estimated by difference. Portions of the solutions containing the oxidation products were used also for measuring the optical rotations and the hydrogen-ion concentrations. The results obtained from a and fi-d glucose and the equilibrium solution are given in Tables 1 and 2. Table 1. Comparison of the oxidation pi oducts with the delta lactone a-d-glucose, oxidation 0-d-glucose, oxi lation d-glucose in equilibrium, 85.5 per cent com- 97.5 per cent complete oxidation 77.2 per cent Gluconic delta lactone plete in 20 minutes in 3 minutes complete in 5 minutes pHi 2 pH pH ! T 120 pH MS H n° MS Hd Minutes Minutes Minutes Minutes 26 50.6 5.1 13 62.4 4.8 14 65.8 4.9 13 61.7 4.6 30 49.7 4.5 15 61.9 4.5 15 65.6 4.7 15 61.4 4.3 40 47.8 3.9 25 60.1 3.6 30 61.5 3.7 25 59.9 3.5 90 39.8 3.3 40 57.3 3.2 45 58.0 3.6 40 57.1 3.1 210 26.8 3.0 60 52.0 2.9 73 52.4 3.3 60 53.8 2.8 355 18.0 2.9 150 27.8 2.5 133 42.3 3.0 150 32.0 2.4 Hours 24 12.0 305 10.1 2.5 338 24.1 2.7 315 14.6 2.4 Hours Hours Hours 24 7.1 22 19.4 24 7.5 1 Measurements made at 25° C. in solutions containing COj and BaBra. Table 2. Saponification of the oxidation products in solutions buffered with BaCOz 1 and C02 at 25° C. Product from equi- Product from Product from Gluconic delta librium solution a-d-glucose 0-d-glucose lactone of d-glucose Per cent Per cent Per cent Per cent of prod- of prod- of prod- of prod- Time uct not Time uct not Time uct not Time uct not saponi- saponi- saponi- saponi- fied fied fied fied Minutes Minutes Minutes Minutes 25 87.4 7 95.8 9 87.1 7.5 93.6 85 63.0 65 70.8 67 63.3 95 62.8 205 40.2 186 43.7 181 36.9 186 44.8 326 29.5 300 26.3 318 21.5 300 20.65 1,440 10.7 1,440 0.7 1,440 2.85 1, 440 00 1 The measurements were made with portions of the solutions given in Table 1.